The vacuolar NHX-type Na(+)/H(+) exchangers play a key role in salt tolerance in plants. However, little is known about the Na(+)/H(+) exchangers in the salt-resistant tree, Populus euphratica. In this study, we identified six putative vacuolar Na(+)/H(+) exchanger genes from P. euphratica, designated as PeNHX1-6. Real-time polymerase chain reaction indicated that the PeNHX1/3/6 transcripts were abundant compared with the other three PeNHX genes in the three tissues (roots, stems and leaves) examined. After NaCl treatment for 6 h, the transcript levels of PeNHX1-6 were upregulated in the roots. To address the function of PeNHX1-6, complementation studies were performed with the salt-sensitive yeast mutant strain R100, which lacks activity of the endosomal Na(+)/H(+) antiporter NHX1. The results showed that PeNHX1-6 compensates, at least in part, for the function of yeast NHX1. Moreover, PeNHX3 was targeted to the tonoplast when transiently expressed in onion. Together, these results suggest that PeNHX1-6 function as vacuolar Na(+)/H(+) exchangers and that PeNHX products play an important role in the salt resistance of P. euphratica.